Human cytomegalovirus infection: new methods for virological diagnosis

Recently developed methods have greatly increased the sensitivity and speed of virological diagnosis of cytomegalovirus infection. Virus can be detected in infected cell cultures within 24 or 48 hours of specimen inoculation by using monoclonal antibodies to immediate-early antigens in immunocytochemistry procedures or DNA sequences in hybridisation in situ assays. CMV antigens can also be detected directly in infected cells within clinical specimens. An early antigen can be visualized in nuclei of circulating leukocytes from viremic patients. DNA hybridization is used for CMV analysis in Dot-blot, Southern-blot and in situ hybridization assays. DNA amplification, by polymerase chain reaction (PCR), has proven to be a very sensitive method for diagnosis of CMV infection and should be useful for investigation of CMV pathogenesis and latency. Serologic assays such as ELISA and latex agglutination assays are accurate for screening donors and recipients of blood and organ or marrow graft. Studies of viral protein epitopes recognized by human sera are in progress.     

A dual role for endothelial cells in cytomegalovirus infection? A study of cytomegalovirus infection in a series of rat endothelial cell lines

Several clinical findings point to the involvement of microvascular endothelial cells in cytomegalovirus-related pathology. In this study the interactions of cytomegalovirus (CMV) with microvascular endothelial cells was investigated in an in vitro rat model. A series of rat endothelial cell lines, considered representative for the heterogeneity of heart microvascular endothelium in vivo, were infected with rat CMV (RCMV). The course of infection and production of infectious virus were examined using immunofluorescence staining and plaque titration assays, and was compared with infection of fully permissive rat fibroblasts. These endothelial cell lines displayed differences in susceptibility to CMV infection. Two endothelial cell lines (RHEC 50 and 191) were practically non-permissive, while four endothelial cell lines (RHEC 3, 10, 11 and 116) were partly permissive for CMV infection. In contrast to CMV infection in fibroblasts, only limited infection of the permissive endothelial cell lines was observed without spreading of CMV infection through the monolayer, although infectious virus was produced. Detachment of infected endothelial cells and recovery of the monolayer with time was observed. The detached endothelial cells were able to transmit CMV infection to fibroblast monolayers, but not to endothelial monolayers. Our in vitro results demonstrate differences in permissiveness for RCMV between the series of rat endothelial cell lines, which is suggestive for endothelial heterogeneity to CMV infection in vivo. Our findings indicate that endothelial cells are relatively resistant to CMV infection and that, upon infection, the endothelial monolayer may dispose of the virus via detachment of the infected cells. This points to a dual role for the endothelium in CMV infection in vivo: a barrier for CMV infection (by the endothelial monolayer) on the one hand and spreading of CMV infection (by detached infected cells) on the other hand.     

Cell-mediated HTLV-I infection of a cervix-derived epithelial cell line

There is considerable evidence that sexual transmission of human T-cell leukemia virus-I (HTLV-I) is mediated by virus-infected lymphocytes in genital tract secretions. However, it is not clear whether infection occurs through lesions in the genital tract epithelium or takes place via an intact epithelium. We have carried out experiments to test the hypothesis that sexual transmission of HTLV-I is initiated by lymphocyte-mediated infection of intact genital tract epithelia. To examine this question we added either free virus or HTLV-I producing MT-2 cells to cultures of a cervix-derived epithelial cell line, MS751. Although free virus did not infect MS751 cells, MS751 cells which had been coincubated with MT-2 cells became infected. These cultures produced about 50 pg/ml of HTLV-I p24 antigen per 10(6) cells over a 24 h period on the sixth day following exposure to donor T-cells. Proviral DNA could be detected in target MS751 epithelial cells by PCR. Infection of epithelia could be blocked, in a dose-dependent manner, by the sulfated polysaccharides dextran sulfate, heparin, and fucoidan, and by the enzymes fucosidase and mannosidase, but not by a number of other agents that were tested. Since MT-2 cells were observed to attach to the epithelial monolayer, we examined the ability of agents to inhibit adhesion. Adherence was inhibited by the same agents that inhibited infection. Based on these findings, we hypothesize that sexual transmission of HTLV-I may involve lymphocyte-mediated infection of genital tract epithelia and that lymphocyte adhesion to the epithelium is a critical event in transmission of HTLV-I. We speculate that a sugar moiety on the epithelium, possibly mannose or fucose, may be involved in adhesion of T-cells to epithelial cells. As sulfated polysaccharides block both adhesion and productive infection of the epithelium, these compounds might be used as active ingredients in a vaginal formulation to help prevent HTLV-I transmission.     

Human herpesvirus 6 reactivation is associated with cytomegalovirus infection and syndromes in kidney transplant recipients at risk for primary cytomegalovirus infection

A potential association between human herpesvirus 6 (HHV-6) and cytomegalovirus (CMV) following kidney transplantation was explored by retrospectively testing serial serum specimens for HHV-6 IgG and IgM antibody. HHV-6 reactivation occurred in 35 (66%) of 53 transplant recipients. Fungal or parasitic opportunistic infections, graft rejection or loss, and mortality were not associated with HHV-6 reactivation. HHV-6 reactivation was associated with primary CMV infection (P=.001) and CMV syndrome (P=.003) and with trends for CMV-related hepatitis (P=.095), CMV-related neutropenia (P=.104), and serious CMV disease (P=.085). After controlling for CMV immune globulin (CMVIG) prophylaxis, the association between HHV-6 reactivation and primary CMV infection and syndrome remained significant (P=.002 and 0.006, respectively). The reduction in CMV syndrome among those receiving CMVIG prophylaxis remained significant (P=.007) after controlling for HHV-6 reactivation. HHV-6 reactivation in kidney transplant recipients at risk for primary CMV infection is associated with CMV infection and CMV-related disease, and these effects are independent of CMVIG prophylaxis.     

Localization of Chlamydia trachomatis heat shock proteins 60 and 70 during infection of a human endometrial epithelial cell line in vitro

Unlike chlamydial lipopolysaccharide, which is released from the developing inclusion to the surface of infected genital epithelial cells, both Chlamydia trachomatis heat shock protein (hsp) 60 and 70 antigens remained confined within the inclusion during the course of the chlamydial developmental cycle. Exposure of the infected cells to penicillin to induce a persistent infection or to a lipophilic microbicide did not potentiate secretion or exocytosis of the chlamydial hsp.     

Transformation in vitro of a nontumorigenic rat urothelial cell line by hydrogen peroxide

Chronic infection/inflammation of the urinary tract is a significant risk factor for the development of bladder cancer. The present study examined the hypothesis that hydrogen peroxide (H202) and cytokines released during inflammation are involved in the enhancement of bladder carcinogenesis. Using growth in soft agar and tumorigenicity in athymic nude mice as indices of transformation, we examined the effect of H202 and cytokines on the enhancement of N-methyl-N-nitrosourea (MNU)-initiated transformation of MYP3 cells, an anchorage-dependent nontumorigenic rat bladder epithelial cell line. MYP3 cells pretreated with or without MNU were exposed to H202 (0.001 to 0.1 mM) daily for 1 week in monolayer culture and were then tested for growth in soft agar. A marked increase in colony numbers was observed in the cells that were MNU-initiated and exposed to H202 (P < 0.01). Furthermore, H202 exposure alone at 0.01 mM or 0.1 mM caused colony formation in soft agar. The transformants induced by MNU plus H202 or H202 alone formed high-grade transitional cell carcinomas when injected into nude mice. The growth of these transformants was stimulated by several cytokines (interleukin 1alpha, interleukin 6, and tumor necrosis factor-alpha) better than the parental cells both on a plastic surface and in soft agar. Our results indicate that H202 causes genetic change(s) to induce tumorigenic conversion in urothelial cells and that the transformants are stimulated to grow because of their selective response to several cytokines. We suggest that these mechanisms may be involved in the in vivo carcinogenesis associated with chronic urinary tract infection.     

Human cytomegalovirus in blood of immunocompetent persons during primary infection: prognostic implications for pregnancy

Human cytomegalovirus (HCMV) was investigated in peripheral blood leukocytes (PBL) of 52 immunocompetent patients (40 pregnant women) with primary HCMV infection by quantitative determination of pp65 antigenemia, viremia, and leukoDNAemia. pp65 antigenemia was detected in 12 (57.1%) of 21, 4 (25%) of 16, and 0 of 10 patients examined 1, 2, and 3 months after onset, respectively. Viremia was detected in 5 (26.3%) of 19 patients during the first month only. LeukoDNAemia was detected in 20 of 20, 17 (89.5%) of 19, and 9 (47.3%) of 19 patients tested 1, 2, and 3 months after onset, respectively. Four (26.6%) of 15 patients were still DNAemia-positive at 4-6 months, whereas none were positive at >6 months. HCMV was not detected in PBL of 20 HCMV-immune donors or of 9 seropositive subjects with recurrent infection. Virus levels were low by all assays and did not correlate with clinical course of infection, intrauterine transmission, or severity of outcome. Invasive procedures in the presence of maternal leukoDNAemia did not seem to interfere with vertical transmission of HCMV infection.     

In vivo and in vitro erythropoietin activities in cultures of a hepatocellular carcinoma cell line

The present studies were undertaken to characterize erythropoietin (Ep) production in an Ep-producing hepatocellular carcinoma (Hep3B) cell line. Hep3B cells which had been maintained in culture were transplanted under the renal capsule and subcutaneously in nude mice. The Hep3B xenograft doubling time is approximately 7 days. The mean hematocrit value of the Hep3B tumor-bearing nude mice was 33.2 +/- 1.1% (n = 8), which was significantly lower than that of control nongrafted nude mice (40.8 +/- 1.7%, n = 5). The Hep3B tumor-bearing nude mice showed significantly higher Ep levels in the sera (37.5 +/- 5.5 munits/ml, n = 8) than control nude mice (13.5 +/- 2.7 munits/ml, n = 5). Ep levels in the sera were correlated (R = 0.714) with the total Ep in the tumor extracts, whereas no Ep was detectable in any of the kidney extracts. On the other hand, an inverse linear relationship (R = -0.811) between the hematocrit values and Ep levels in the sera was demonstrated in the Hep3B tumor-bearing nude mice. The Hep3B cells recultured after growing in the nude mice were capable of enhancing Ep production in response to hypoxia, very similar to the original Hep3B cells which had been maintained in culture during the same time period. In addition, 15-methyl-prostaglandin E1 at a concentration range of 4-400 ng/ml produced significant increases in Ep secretion and cAMP accumulation in Hep3B cultures under hypoxic conditions (1% O2). The Ep produced by Hep3B cells expressed 3.7 times higher in vitro bioactivity than immunoactivity. The bioactivity of Hep3B Ep was completely neutralized by an antibody to highly purified human recombinant Ep. In contrast, the in vivo bioactivity of the Hep3B Ep was less than one tenth of its immunoactivity. These results indicate that the Hep3B tumor-bearing nude mice and the in vitro Hep3B culture system may provide a reproducible model system which should be useful for studies of the mechanism of Ep production.     

Differing infection patterns of dengue and yellow fever viruses in a human hepatoma cell line

Dengue (DEN) and yellow fever (YF) viruses are responsible for human diseases with symptoms ranging from mild fever to hepatitis and/or hemorrhages. Whereas DEN virus typically induces only limited foci of necrosis in the liver, YF virus infection is characterized by devastating lesions. In a human hepatoma cell line (HepG2), the kinetics of DEN and YF virus replication and release from the cells and the nature of host cell response to viral infection were compared. DEN virus infection was characterized by the early appearance of intracellular viral antigens, major ultrastructural cytopathic changes as early as 32 h after infection, extensive apoptotic cell death, and a low production of infectious particles. In contrast, YF virus grew exponentially to high titers and induced cytopathic changes only 72 h after infection. Differences between the infection processes of the two viruses observed in the hepatoma cell line may explain the different liver pathologies.     

Expression of the retinoblastoma (RB) tumor suppressor gene inhibits tumor cell invasion in vitro

To determine if replacement of the retinoblastoma (RB) tumor suppressor gene could inhibit invasion of RB-defective tumor cells, the capacity of tumor cells to migrate or invade was quantitated by the Boyden chamber assay. The studies were done in a diverse group of stable RB-reconstituted human tumor cell lines, including those derived from the osteosarcoma and carcinomas of the lung, breast and bladder. The expression of the exogenous wild-type RB protein in these tumor cell lines was driven by either a constitutively active promoter or an inducible promoter. It was found that significantly more tumor cells from the parental RB-defective cell lines and the RB revertants than from the RB-reconstituted RB+ cell lines penetrated through the Matrigel (P<0.001, two-tailed t-test), although both RB+ and RB- cells migrated at approximately the same rate on uncoated polycarbonate filters in the Boyden chambers. Of note, the inhibition of invasiveness of various RB-defective tumor cells by RB replacement was apparently well correlated with suppression of their tumorigenicity in vivo. In contrast, although either functional RB or p53 re-expression effectively suppressed tumor formation in nude mice of the RB-/p53null osteosarcoma cell line, Saos-2, replacement of the wild-type p53 gene had much less impact on their invasiveness as compared to the RB gene. These studies provided an insight into the broader biological basis of the RB-mediated tumor suppression in RB-defective tumor cells.     

Initiation of human cytomegalovirus infection requires initial interaction with cell surface heparan sulfate

In this report, we demonstrate that the initial event in human cytomegalovirus (HCMV) infection is attachment to extracellular heparan sulfate. Further, this interaction is important for initiation of infection in fibroblast cells. Using microbinding assays to specifically monitor virus attachment as well as plaque titration assays to measure infectivity, we found that heparin competition as well as enzymatic digestion of cells with heparinase blocked virus attachment, initiation of immediate-early gene expression and infectivity. Other major glycosaminoglycans were found not to be involved in HCMV attachment and infectivity. In addition, HCMV was unable to attach to mutant derivatives of Chinese hamster ovary cells deficient in synthesis of heparan sulfate proteoglycans. Basic fibroblast growth factor, which requires initial interaction with extracellular heparin prior to binding to its high affinity receptor, also inhibited HCMV attachment to cells. Time-course experiments revealed that the initial HCMV binding was sensitive to heparin competition (10 micrograms/ml) or 0.75 M salt washes. The initial heparin-dissociable binding converted rapidly to high affinity (heparin resistant) HCMV attachment. These data suggest that sequential receptor interactions may mediate HCMV adsorption to cells. Heparin affinity chromatography revealed that multiple HCMV envelope glycoproteins, including gB, are capable of binding to heparin.     

Electrical properties of Y-79 cells, a multipotent line of human retinoblastoma

1. Whole-cell and perforated-patch tight-seal recording techniques were used to characterize the voltage-dependent membrane conductances of the Y-79 cells, a human retinoblastoma line composed of pluripotential retinal precursor cells. 2. Membrane resistance and capacitance were measured under current clamp, yielding approximate average values of 1.8 G omega and 26 pF, respectively. The cells are electrically excitable, and depolarization above -20 mV triggers slow action potentials. 3. Step depolarization of the membrane under voltage clamp elicits a high-threshold transient inward current, followed by a sustained, larger outward current. The outward current is carried by potassium ions, as determined by its susceptibility to blockage by K-channel antagonists [tetraethylammonium (TEA), Cs, and 4-aminopyridine (4-AP)] and insensitivity to reduction of external chloride concentration. 4. The isolated inward current displayed some unusual properties: its amplitude is directly related to extracellular calcium concentration, and replacement of calcium by magnesium completely abolishes it. However, none of the calcium channel antagonists tested (cadmium, nickel, nifedipine, and amiloride) exerted a substantial blockage. In addition, removal of external sodium or superfusion with tetrodotoxin significantly reduce the size of this current. 5. A single voltage-dependent conductance appears to underlie the inward current, because a variety of manipulations, such as changes in the holding potential, in the extracellular concentration of calcium or sodium, or superfusion with tetrodotoxin, failed to reveal the presence of kinetically distinct components. 6. The results suggest that a single voltage-dependent conductance mechanism underlies the depolarization-activated inward current in Y-79 cells. This channel appears to be primarily permeable to calcium, but with a significant contribution by sodium ions. Its functioning appears to be modulated by extracellular calcium.     

Human lymphoblastoid cell line producing specific antibody against Rh-antigen D

A lymphoblastoid cell line producing specific anti-Rh antibody against D determinant was established by Epstein-Barr virus (EBV) infection. The lymphocytes were from an immunized male donor with high titre of anti-D antibody. The cells were preselected by rosetting them with Rh-positive erythrocytes before EBV infection. The cell line produced specific antibody of IgG class, namely IgG1 subclass, but it also produced nonspecific IgM and IgG. By rerosetting the cell line, the specific antibody-producing population could be enriched and the antibody titre increased in the culture supernatant to the level of immune sera.     

A patient with a cytomegalovirus infection and multiple stomach ulcers

In a 64-year-old, previously healthy woman an upper gastrointestinal endoscopy was performed because of serious dyspeptic complaints during fourteen days. Multiple fundic gastric ulcers were found without Helicobacter pylori and the patient did not take non-steroidal anti-inflammatory drugs (NSAIDs). The diagnosis was primary cytomegalovirus infection, based on the demonstration of infected cells in the biopsy specimens, using specific monoclonal anti-cytomegalovirus antibodies.     

Bone abnormalities and fetal cytomegalovirus infection

BACKGROUND: Viruses, such as those of congenital rubella and cytomegalovirus infection, have been associated with bone lesions of rarefaction. CASE REPORT: A full-term neonate was admitted at bith (birth weight: 1,630 mg; head circumference: 29 cm) for hypotrophy. A severe growth retardation had been diagnosed at 32 weeks of gestation. The fetal karyotype was normal. Search for maternal toxoplasmosis, syphilis and rubella was negative. The neonate had a complex heart defect; skeletal X-ray films showed metaphyseal transverse bands of rarefaction of the long bones and longitudinally streaked band of sclerosis and rarefaction at the ends of the femora. Urine culture was highly positive for CMV while CMV IgM were negative and CMV IgG highly positive in serum. CONCLUSIONS: Neonatal bone changes usually seen in congenital rubella infection may also be observed in CMV infection.     

Diagnosis and treatment of cytomegalovirus infection

During the period 1979-1991, Salmonella Typhimurium DT 204c was the cause of a major epidemic of salmonellosis in calves in the UK. Plasmid profile analysis of DT 204c isolates from England and Wales commenced in 1986 and isolates from all subsequent incidents were examined by this technique. Forty-three different plasmid profile types (PPTs) were detected, of which the commonest, designated type E, constituted 44.6-80.2% of the annual incidents during the study period. Some PPTs, e.g., F and P, were detected throughout most years of the study, whereas PPTs O and 6 persisted for short periods. Until 1984, most isolates were resistant to neomycin, but the subsequent predominant PP type E was sensitive to this antibacterial agent. It was concluded that during the epidemic there was an evolution of new genotypes, of which only some persisted; again, antibacterial resistance genes may be acquired or lost. The study demonstrated the value of PP typing for epidemiological studies.     

Prenatal diagnosis of congenital cytomegalovirus infection

We report here the results of a study on the prenatal diagnosis of congenital cytomegalovirus (CMV) infection. The study was carried out by both PCR and virus isolation from amniotic fluid (AF) for 82 pregnant women at risk of transmitting CMV for the detection of (i) seroconversion to CMV immunoglobulin G (IgG) positivity during the first trimester of pregnancy, (ii) symptomatic CMV infection in the mother during the first trimester of pregnancy or intrauterine growth retardation detected by ultrasound or abnormal ultrasonographic findings suggestive of fetal infections, and (iii) seropositivity for CMV-specific IgM. For 50 women, fetal blood (FB) was also obtained and tests for antigenemia and PCR were performed. The results indicate that AF is better than FB for the prenatal diagnosis of CMV infection. PCR with AF has a sensitivity (SNS) of 100%, a specificity (SPE) of 83.3%, a positive predictive value (PPV) of 40%, and a negative predictive value (NPV) of 100%; rapid virus isolation with the same material has an SNS of 50%, an SPE of 100%, a PPV of 100%, and an NPV of 94.7%. Fewer than 10% of the women positive for IgM by enzyme immunoassay (EIA) had a congenitally infected fetus or newborn infant. When EIA IgM positivity was confirmed by Western blotting (WB) and the WB profile was considered, the percent transmission detected among women with an "at-risk" profile was higher than that observed among IgM-positive women and was the same as that among women who seroconverted during the first trimester of pregnancy (transmission rates of 29 and 25%, respectively).